This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Abstract

Background

Programmed death-1 (PD-1, Pdcd1)-deficient mice develop different types of autoimmune diseases depending on the mouse
strain but its role in uterus development has not been reported.

Methods

In this study, the expression of PD-1 and its ligands, PD-L1 and PD-L2, in uterine
tissues from aged WT mice in a 129svEv-Brd background was analyzed by immunohistochemistry
and the uterine morphology between WT and PD-1-/- mice was compared by hematoxylin and eosin staining.

Results

The aged PD-1-/- female mice in a 129svEv-Brd rather than Balb/c background develop endometrial hyperplasia.
H&E staining showed an increase in the number of glands, neovascularization and an
extremely large luminal cavity in aged PD-1-/- uteri. Immunohistochemical assay showed that the expression of PD-1 was observed
in glandular/luminal epithelium and cells infiltrating the stroma. Fluorescent double
staining demonstrated that PD-1 expresses on CD68+ macrophages, CD3+ T cells, CD16+ monocytes, CD56+ NK cells and CK-18+ epithelial cells, respectively. Additionally, PD-1 co-expresses with vascular endothelial
growth factor (VEGF), and PD-1 deficiency resulted in an accumulation of glandular/luminal
epithelium derived VEGF, which accelerates the expression of the proliferation-associated
protein, proliferating cell nuclear antigen (PCNA), and thus potentially lead to epithelial
proliferation in aged PD-1-/- uteri.

Conclusions

These findings showed that PD-1 deficiency augments luminal epithelial cell proliferation
probably through induced VEGF secretion, suggesting PD-1 plays an important role in
controlling the growth and differentiation of the uterine epithelium.

Virtual Slides

Keywords:

Background

Co-signaling by B7/CD28 family members regulates the initiation, maintenance, and
termination of immune responses. Programmed death-1 (PD-1) is an inhibitory receptor
expressed on activated T cells, B cells and myeloid cells [1]. PD-1 deficiency (PD-1-/-) causes lupus-like glomerulonephritis and arthritis in C57BL/6 mice [2,3], autoimmune dilated cardiomyopathy (DCM) and gastritis in BALB/c mice [4,5], acute type 1 diabetes mellitus (T1DM) in nonobese diabetic (NOD) mice [6], and lethal myocarditis in MRL mice [7]. In humans, polymorphisms in the PD-1 gene have been associated with susceptibility
to systemic lupus erythematosus [8], type I diabetes [9], multiple sclerosis [10], and rheumatoid arthritis [11]. Additionally, PD-1-/- mice in a 129svEv-Brd background were also more susceptible to the development of
experimental autoimmune encephalomyelitis (EAE) [12]. Nevertheless, the organ development regulated by PD-1 signal is still under investigation.

PD-L1 (B7-H1) and PD-L2 (B7-DC), two immunoregulatory molecules belonging to the B7
family, were identified as the ligands for PD-1 [13,14]. The expression of PD-L1 has been detected not only in lymphoid organs but also in
nonlymphoid tissues and was enhanced in several types of tumor cells under inflammation
conditions, suggesting that PD-L1 might regulate lymphocyte function at sites of inflammation
[15]. The expression of PD-L2, however, was restricted in activated dendritic cells (DCs),
macrophages, monocytes and T cells [16].

The expression, anatomic distribution and potential role for PD-1/PD-Ls in uterine
development have not been investigated. We here showed that aged PD-1-deficient female
mice in a 129svEv-Brd background develop endometrial hyperplasia. This effect potentially
reflects the induction VEGF secretion from epithelial cells upon PD-1 signaling deficiency.

Methods

Ethics statement

All experiments were approved and conducted in accordance with the guidelines of the
Animal Care and Use Committee of the Third Military Medical University. All efforts
were made to minimize animal suffering.

Mice

PD-1-deficient mice (Background: 129svEv-Brd) were kindly provided by Dr. Laura L.
Carter (Inflammation Department, Wyeth Research, Cambridge, MA, USA). Prof. T. Honjo
(Department of Immunology and Genomic Medicine, Kyoto University, Japan) kindly gave
us the PD-1-KO-N10 mice (strain: BALB/cJ). The WT control mice were purchased from
the Animal Center of Beijing University School of Medicine. All mice were maintained
in micro-isolator cages and housed in the animal colony at the Animal Center, Third
Military Medical University, and standard laboratory chow diet and water was supplied.

Cell count and statistical analysis

The proportion of PCNA-positive nuclei in the glandular epithelium was determined
through image analysis of the histological sections. Photomicrographs were captured
and analyzed using Image Pro-Plus 5.0 software (Media Cybernetics, Silver Spring,
MD) [18]. The number of PCNA+ nuclei per high-power field was counted. The data were analyzed using GraphPad Prism
4.03 software. An unpaired Student t test (two-tailed) was used to assess comparisons of PCNA+ nuclei between PD-1-/- and WT uteri. A p value <0.05 was considered statistically significant different.

Results

Changes in the gross anatomy and morphology of the uteri in aged PD-1-/- female mice

The WT and PD-1-/- mice at different ages were sacrificed, and the morphology of several organs was
compared. An interesting finding is the uteri from PD-1-/- female mice in the 129svEv-Brd rather Balb/c background are much larger than that
of the WT littermates at 2, 2.5 and 3 years of age (Figure 1B and C), indicating that the aged PD-1-/- female mice developed endometrial hyperplasia. Nevertheless, the uteri from young
(for example, 1-year-old) PD-1-/- and WT mice in 129svEv-Brd background were comparable (Figure 1A). H&E staining showed that there were numerous glands (Figure 1E) and neovascularizations (Figure 1F) in the aged PD-1-/- uteri (129svEv-Brd background), as compared to WT controls at 2 years of age (Figure 1D). These findings suggested that endometrial hyperplasia was developed in aged PD-1-/- female mice in a 129svEv-Brd background.

Figure 1.Gross anatomic and histological comparison of uteri between PD-1-/- and WT mice. Gross anatomy of uteri between PD-1-/- and WT mice (129svEv-Brd background) at (A) 1 year and (B) 2 years of age, results showed that uteri from PD-1-/- mice in 2-year old are more larger than WT littermates. One of represents five mice
was shown. (C) Gross anatomy of uteri from PD-1-/- and WT mice on Balb/c background at 2-year age was compared, results showed that
uteri from PD-1-/- mice were similar to than from WT littermates. One of represents five mice was shown.
Histology of uteri from WT (D) and PD-1-/- mice (E and F) in a 129svEv-Brd background at 2-year age was compared by using H&E staining. Star
indicated glands and arrow showed neovascularization; D-F, Scale bar = 20 μm; N = 5 of each group.

PD-1 deficiency resulted in epithelial cell proliferation

To analyze the pathogenesis of endometrial hyperplasia in aged PD-1-/- female mice, the expression of the proliferation-associated protein, proliferating
cell nuclear antigen (PCNA), was analyzed in aged uteri between PD-1-/- and WT mice in 129svEv-Brd background. Immunofluorescent staining revealed the presence
of PCNA+ cells in the uteri, and PCNA expression was primarily detected in luminal/glandular
epithelium (Figure 2A). The PCNA+ nuclei were counted and results showed that 42.1 ± 3.2% of luminal/glandular epithelial
cells in PD-1-/- uteri were positive for PCNA, while only 21.4 ± 1.2% of the cells in WT uteri showed
nuclei positive for PCNA (Figure 2B), suggesting that PD-1 deficiency triggers epithelial cell proliferation in the
aged uteri.

The expression of the PD-1ligands, PD-L1 and PD-L2, in the aged uteri from WT mice
was also detected by immunohistochemistry. Similar to PD-1, the expression of PD-L1
was also observed in glandular/luminal epithelium (Figure 4B). Cells infiltrating the stroma and some endothelial cells were also positive for
PD-L1 (Figure 4C). In contrast, the expression of PD-L2 was absent in blood endothelium (Figure 4D) but present in glandular/luminal endothelium and cells infiltrating the stroma
(Figure 4E and F).

Discussion

The disruption of the PD-1 signal leads to the breakdown of peripheral tolerance and
the initiation of autoimmunity like dilated cardiomyopathy. This effect is due to
PD-1 negatively controls T cell receptor (TCR) signaling [19]. Recently, it was shown that PD-1 deficiency accelerates microRNA-21 (miR-21) overexpression,
thus lead to cell proliferation through the enhanced expression of programmed cell
death 4 (PDCD4) [20]. Here, we provided the first evidence that endometrial hyperplasia was developed
in aged PD-1-/- female mice in a 129svEv-Brd rather than Balb/c background (Figure 1B), suggesting PD-1 plays an important role in the growth and differentiation of the
uterine epithelium in129svEv-Brd mice. Our results further reflect that the development
of disease mediated by PD-1 signals in animal models is strain restricted.

The expression of PD-1 has been reported on T cells, natural killer T cells, B cells
and monocytes, and this expression was enhanced through stimulation with inflammatory
factors, such as TNF-α and IFN-γ. However, PD-L1 and PD-L2, the two PD-1ligands, shows
different expression patterns [21]. Here, we showed that the expression of PD-1 was observed on glandular and luminal
epithelium (Figure 3B) and cells infiltrated in stroma (Figure 3C). Additionally, PD-1 was also detected to be expressed on CD68+ macrophages, CD3+ T cells, CD16+ monocytes, CD56+ NK cells and CK-18+ epithelial cells, but it was absent on CD31+ endothelial cells, as detected by immunofluorescent double staining (Figure 3D). Additionally, the expression of PD-L1 and PD-L2 was also detected on luminal epithelium
and infiltrating cells within the aged uterine tissues (Figure 4). Due to strong neovascularization (Figure 1F) and higher level of PCNA+ cells were seen in aged PD-1-/- uteri, suggesting strong cell proliferation is in progress in PD-1-/- uteri (Figure 2). These combine data suggest that local PD-1/PD-Ls signal probably controls glandular/luminal
epithelial biofunction, like cell proliferation and neovascularization. Indeed, the
expression of PD-1 on the tubular epithelium of murine Adriamycin nephropathy (AN)
has been reported previously, and blockade of PD-1 worsened progressive renal histopathological
and functional injury in murine AN [22]. Taken together, our results suggested that PD-1 is not only expressed on immune
cells but also on nonlymphoid tissues, and uterine local PD-1/PD-Ls signal probably
directly inhibits glandular/luminal epithelial cell proliferation and neovascularization.

To analyze other potential molecular mechanisms that involve in the growth and differentiation
of the uterine epithelium in aged PD-1-/- mice, the secretion of VEGF, which stimulate endothelial and epithelial cell proliferation
through its receptor, VEGFR, was compared in aged uteri between PD-1-/- and WT mice. An interesting finding is that glandular/luminal epithelium derived-VEGF
in aged uteri from PD-1-/- mice was augmented dramatically (Figure 5C), thereby potentially promotes cell proliferation via its receptor VEGFR and thus resulted in accelerating neovascularization (Figure 1F). Additionally, VEGF in uteri was also co-expresses with PD-1 (Figure 5D), suggesting that PD-1 signaling inhibits epithelial cell proliferation potentially
through a reduction of VEGF secretion, in addition to direct prevents epithelial proliferation
by cross-reacts with PD-Ls. However, the expression of PTEN, a tumor suppress gene,
were significantly higher in cyclical endometrium than in atypical hyperplasia and
endometrioid carcinoma, indicated that PTEN involves in the pathogenesis of endometrial
hyperplasia [23]. On the other hand, CyclinD1, a cell -cycle regulator, exhibited a promising potential
to predict the prognosis of patients with endometrial carcinoma [24]. Whether the transcription of PTEN or CyclinD1 is also controlled by PD-1/PD-Ls need
further investigation.

Conclusion

PD-1 deficiency augments luminal epithelial cell proliferation and neovascularization
in aged uteri, suggesting that PD-1 plays an important role in the organization, growth
and differentiation of the uterine epithelium.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

GG is responsible for immunohistochemistry, HL participated in the discussion for
histological diagnosis and manuscript preparation. DC was responsible for immunofluoresent
double staininge and YC is preparing the manuscript. All authors read and approved
the final manuscript.

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of
China (NSFC No. 8122223 and No. 61141012).